Molecular studies of the calmodulin (CaM)-dependent protein phosphatase, calcineurin (CN), have identified 3 mammalian genes for the catalytic (A) subunit and determined their chromosomal localization in humans. Characterization of A subunit genes in many phyla indicate that mammalian forms arose from an ancestral line that includes the filamentous fungi (Neurospora, Aspergillus) and suggests that gene divergence occurred after the avian/reptilian branch point. Two genes for the regulatory (B) subunit were cloned, one of which is co-expressed with an isoform of the A subunit during testicular development; these two subunits may comprise an isoenzyme that is associated with the sperm flagellum. Biochemical studies with recombinant proteins, expressed in bacteria and in insect cells, show that the B subunit isoforms can associate interchangeably with different A subunits and with a fungal catalytic subunit. These data indicate functional conservation of the B subunit interaction domain, which has now been delineated in mapping studies. An important biological role for CN was established in T-cell regulation. In human cells transfected with a constitutively-active mutant of the A subunit, the interleukin (IL)-2 promoter was activated synergistically by phorbol esters; this suggests a role for CN in T-cell receptor-mediated signaling events that regulate IL- 2 gene transcription. These cells also were resistant to the immunosuppressants, FK-506 and cyclosporin A, in agreement with recent findings that CN forms complexes with the cytosolic drug receptors ("immunophilins"); thus, CN is the primary target of these drugs in T- cells. A CaM-regulated phosphodiesterase (PDE) was cloned from murine brain by PCR amplification of mRNA from microdissected cerebellar Purkinje cell layers. In situ hybridization and Northern blot analysis indicate high expression of PDE mRNA in the striatum, suggesting a role in control of dopamine-mediated cyclic AMP pathways. Peptide microsequencing was completed on a novel 36 kDa CaM-binding protein isolated from brian, showing no homology to any mammlaina proteins. Western blot analysis with anti-peptide antibodies showed highest amounts in brain, spleen and kidney.